One of the fundamental questions of Cat+-mediated signal transduction is how activation of multiple enzymes by changes in a diffusible second messenger, calcium, leads to temporally and spatially restricted effects. The objective of this research is to examine further the spatial and temporal dynamics of the Cat+-binding protein calmodulin by measuring its diffusion utilizing a combination of biochemical and imaging approaches. Preliminary experiments and previous work (Persechini & Cronk, 1999; Luby-Phelps et al., 1995; Tansey et al., 1994) suggest that calmodulin is limiting in cells. If found true for neurons, this discovery imparts important constraints on how neuronal calmodulin-dependent enzymes are activated and poses profound implications on the role of calmodulin in acting as the detection system for Ca2+ transients to regulate cellular processes: The specific aims of this research project are: 1) to determine the amount of diffusible and bound calmodulin in PC12 cells at differing Ca2+ concentrations using a biochemical approach; 2) to characterize the compartmentalization and localization of calmodulin in PC12 cells and neurons using immunocytochemistry and confocal microscopy; and 3) to determine the diffusion of calmodulin in PC12 cells and neurons using both multiphoton fluorescence photobleaching recovery (MPFPR) and fluorescence correlation spectroscopy (MPFCS). Through the quantitative analysis of diffusion and spatial localization of calmodulin at different levels of Cat+, a more complete understanding between Ca2+ signaling and calmodulin availability in neurons will be elucidated. These studies will provide essential information for understanding how neurons process information through the Ca2+/calmodulin-dependent signaling pathway.